The present invention relates to vectors, such as recombinant vectors; for instance, recombinant viruses, such as poxviruses, e.g., modified poxviruses and to methods of making and using the same. In some embodiments, the invention relates to recombinant avipox viruses, such as canarypox viruses, e.g., ALVAC. The invention further relates to such vectors, e.g., poxviruses, that express gene products, e.g., antigen(s), ORF(s), and/or epitope(s) of interest therefrom, of porcine circovirus 2 (PCV2); to immunological compositions or vaccines. The invention yet further relates to such vectors, e.g., poxviruses, that induce an immune response directed to or against PCV2 gene products and/or PCV2; and, to advantageously, such compositions that are immunological, immunogenic or vaccine compositions and/or confer protective immunity against infection by PCV2. The invention yet further relates to the uses of and methods for making and using such vectors and compositions, as well as intermediates thereof, and said intermediates. And, the invention relates to the products therefrom, e.g., from the uses and methods involving the inventive recombinant or poxvirus, such as antibodies from expression.
Postweaning multisystemic wasting syndrome (PMWS) is a recently recognized disease of young pigs. PMWS is characterized clinically by progressive weight loss and other symptoms such as tachypnea, dyspnea and jaundice. Pathologically, lymphocytic and granulomatous infiltrates, lymphadenopathy, and, more rarely, lymphocytic and granulomatous hepatitis and nephritis have been observed (Clark, 1997; Harding, 1997).
This disease has been described in different European countries as well as in North America. Treatment and prevention of this disease are not currently available.
Several lines of evidence point to porcine circovirus as the etiologic agent of PMWS (Ellis et al., 1998). Circoviruses have been recovered from pigs with PMWS, and antibodies to porcine circovirus have been demonstrated in pigs with the disease.
Circoviruses are single stranded circular DNA viruses found in a range of animal and plant species. Porcine circovirus was originally isolated as a contaminant from a continuous pig kidney cell line. The cell culture isolate has been designated PK-15 (Meehan et al., 1997). More recently, porcine circovirus obtained from pigs with PMWS has been compared to PK-15. Such viruses differ substantially from PK-15 at the nucleotide and protein sequence level, and have been designated PCV2 (Meehan et al., 1998; Hamel et al., 1998).
As many as thirteen open reading frames (ORFs) have been identified in the PCV2 genome (COL1 to COL13 in the French patent application 98 03707). Four of these ORFs share substantial homology with analogous ORFs within the genome of PK-15. ORF1 (Meehan et al., 1998; corresponding to COL4 in the French patent application 98 03707), comprising nt 398-1342 (GenBank accession number AF055392), has the potential to encode a protein with a predicted molecular weight of 37.7 kD. ORF2 (Meehan et al., 1998; corresponding to COL13 in the French patent application 98 03707), comprising nt 1381-1768 joined to 1-314 (GenBank accession number AF055392), may encode a protein with a predicted molecular weight of 27.8 kD. ORF3 (Meehan et al., 1998; corresponding to COL7 in the French patent application 98 03707), comprising nt 1018-704 (GenBank accession number AF055392), may encode a protein with a predicted molecular weight of 11.9 kD. ORF4 (Meehan et al., 1998; corresponding to COL10 in the French patent application 98 03707), comprising nt 912-733 (GenBank accession number AF055392), may encode a protein with a predicted molecular weight of 6.5 kD.
ORF1 of PCV2 is highly homologous (86% identity) to the ORF1 of the PK-15 isolate (Meehan et al., 1998). The ORF1 protein of PK-15 has been partially characterized (Meehan et al., 1997; Mankertz et al., 1998a). It is known to be essential for virus replication, and is probably involved in the viral DNA replication.
Protein sequence identity between the respective ORF2s was lower (66% identity) than that of the ORF1s but each of the ORF2s shared a highly conserved basic N-terminal region, similar to that observed in the N-terminal region of the major structural protein of the avian circovirus chicken anemia virus (CAV) (Meehan et al., 1998). Recently, Mankertz et al. (1998b) has suggested that the ORF2 of the PK-15 isolate (designated ORF 1 in Mankertz et al., 1998b) codes for a capsid protein.
Greater differences were observed between the respective ORF3s and ORF4s of the PK-15 isolate and PCV2. In each case, there was a deletion of the C-terminal region of PCV2 ORF4 and ORF3 compared to the corresponding ORFs present in the genome of the PK-15 isolate. The highest protein sequence homology was observed at the N-terminal regions of both ORF3 and ORF4 (Meehan et al., 1998).
The transcription analysis of the genome of PCV2 has not been published yet. Recent data obtained with the PK-15 isolate indicated that the ORF2 transcript is spliced (Mankertz et al., 1998b).
Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. With the eradication of smallpox, a new role for poxviruses became important, that of a genetically engineered vector for the expression of foreign genes (Panicali and Paoletti, 1982; Paoletti et al., 1984). Genes encoding heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990). A highly attenuated strain of vaccines, designated MVA, has also been used as a vector for poxvirus-based vaccines. Use of MVA is described in U.S. Pat. No. 5,185,146.
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipox viruses. Both fowlpoxvirus (FPV; Taylor et al. 1988a, b) and canarypoxvirus (CPV; Taylor et al., 1991 and 1992) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920""s by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988c). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988c). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).
Other attenuated poxvirus vectors have been prepared by genetic modifications of wild type strains of virus. The NYVAC vector, derived by deletion of specific virulence and host-range genes from the Copenhagen strain of vaccinia (Tartaglia et al., 1992) has proven useful as a recombinant vector in eliciting a protective immune response against an expressed foreign antigen.
Another engineered poxvirus vector is ALVAC, derived from canarypox virus (ALVAC was deposited with American Type Culture Collection, P.O. Box 1549, Manassas, Va. 20108, USA, under the terms of the Budapest Treaty on Nov. 14, 1996, and is designated as Accession Number VR-2547). ALVAC does not productively replicate in non-avian hosts, a characteristic thought to improve its safety profile (Taylor et al., 1991 and 1992). Both ALVAC and NYVAC are BSL-1 vectors.
One approach to the development of a subunit PCV2 vaccine is the use of live viral vectors to express relevant PCV2 ORFs. Recombinant poxviruses can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; 5,174,993; 5,494,807; and 5,505,941, the disclosures of which are incorporated herein by reference. It can thus be appreciated that provision of a PCV2 recombinant poxvirus, and of compositions and products therefrom particularly ALVAC based PCV2 recombinants and compositions and products therefrom, especially such recombinants containing ORFs 1 and/or 2 of PCV2, and compositions and products therefrom would be a highly desirable advance over the current state of technology.
It is therefore an object of this invention to provide compositions and methods for treatment and prophylaxis of infection with PCV2. It is also an object to provide a means to treat or prevent PMWS.
In one aspect, the present invention relates to an antigenic, immunological, immunogenic, or vaccine composition or a therapeutic composition for inducing an antigenic, immunogenic or immunological response in a host animal inoculated with the composition. The composition advantageously includes a carrier or diluent and a recombinant virus, such as a recombinant poxvirus. The recombinant virus or poxvirus contains and expresses an exogenous nucleic acid molecule encoding an ORF, antigen, immunogen, or epitope of interest from PCV2, or a protein that elicits an immunological response against PCV2 or conditions caused by PCV2, such as PMWS. For instance, the recombinant virus can be a modified recombinant virus or poxvirus; for example, such a virus or poxvirus that has inactivated therein virus-encoded genetic functions, e.g., nonessential virus-encoded genetic functions, so that the recombinant virus has attenuated virulence and enhanced safety. And, the invention further provides the viruses used in the composition, as well as methods for making and uses of the composition and virus.
The virus used in the composition according to the present invention is advantageously a poxvirus, particularly a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus and more advantageously, ALVAC. The modified recombinant virus can include, e.g., within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein, e.g., derived from PCV2 ORFs, e.g., PCV2 ORF 1 and/or 2.
In yet another aspect, the present invention relates to an immunogenic composition containing a modified recombinant virus having inactivated nonessential virus-encoded genetic functions so that the recombinant virus has attenuated virulence and enhanced safety. The modified recombinant virus includes, e.g., within a non-essential region of the virus genome, a heterologous DNA sequence which encodes an antigenic protein (e.g., derived from PCV2 ORFs, especially ORFS 1 and/or 2) wherein the composition, when administered to a host, is capable of inducing an immunological response specific to the antigen.
In a still further aspect, the present invention relates to a modified recombinant virus having nonessential virus-encoded genetic functions inactivated therein so that the virus has attenuated virulence, and wherein the modified recombinant virus further contains DNA from a heterologous source, e.g., in a nonessential region of the virus genome. The DNA can code for PCV2 genes such as any or all of PCV2 ORF1, ORF2, ORF3, or ORF4 (Meehan et al., 1998), or epitope(s) of interest therefrom. The genetic functions can be inactivated by deleting an open reading frame encoding a virulence factor or by utilizing naturally host-restricted viruses. The virus used according to the present invention is advantageously a poxvirus, e.g., a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus.
Advantageously, the open reading frame that is deleted from the poxvirus or virus geneome is selected from the group consisting of J2R, B13R+B14R, A26L, A56R, C7Lxe2x88x92K1L, and I4L (by the terminology reported in Goebel et al., 1990); and, the combination thereof. In this respect, the open reading frame comprises a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range gene region or a large subunit, ribonucleotide reductase; or, the combination thereof.
A suitable modified Copenhagen strain of vaccinia virus is identified as NYVAC (Tartaglia et al., 1992), or a vaccinia virus from which has been deleted J2R, B13R+B14R, A26L, A56R, C7Lxe2x88x92K11 and 14L or a thymidine kinase gene, a hemorrhagic region, an A type inclusion body region, a hemagglutinin gene, a host range region, and a large subunit, ribonucleotide reductase (See also U.S. Pat. Nos. 5,364,773, 5,494,807, and 5,762,938, with respect to NYVAC and vectors having additional deletions or inactivations from those of NYVAC that are also useful in the practice of this invention).
Preferably, the poxvirus vector is an ALVAC or, a canarypox virus which was attenuated, for instance, through more than 200 serial passages on chick embryo fibroblasts (Rentschler vaccine strain), a master seed therefrom was subjected to four successive plague purifications under agar from which a plague clone was amplified through five additional passages. (See also U.S. Pat. Nos. 5,756,103 and 5,766,599 with respect to ALVAC and TROVAC (an attenuated fowlpox virus useful in the practice of this invention); and U.S. Pat. Nos. 6,004,777, 5,990,091, 5,770,212, 6,033,904, 5,869,312, 5,382,425, and WO 95/30018, with respect to vectors that also can be used in the practice of this invention, such as vectors having enhanced expression, vectors having functions deleted therefrom and vectors useful with respect to porcine hosts (for instance, vectors useful with porcine hosts can include a poxvirus, including a vaccinia virus, an avipox virus, a canarypox virus, and a swinepox virus), as well as with respect to terms used and teachings herein such as xe2x80x9cimmunogenic compositionxe2x80x9d, xe2x80x9cimmunological compositionxe2x80x9d, xe2x80x9cvaccinexe2x80x9d, and xe2x80x9cepitope of interestxe2x80x9d, and dosages, routes of administration, formulations, adjuvants, and uses for recombinant viruses and expression products therefrom).
The invention in yet a further aspect relates to the product of expression of the inventive recombinant poxvirus and uses therefor, such as to form antigenic, immunological or vaccine compositions for treatment, prevention, diagnosis or testing; and, to DNA from the recombinant poxvirus which is useful in constructing DNA probes and PCR primers.
These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description.